Project Summary: The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing and its regulation. mRNA 3'-end formation, typically involving an endonucleolytic cleavage followed by polyadenylation, is an essential step of eukaryotic gene expression and it significantly impacts many aspects of RNA metabolism, including mRNA stability and translation. In addition, the majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3' ends through alternative polyadenylation (APA). Recent studies have revealed that APA is highly regulated in development and plays an important role in post-transcriptional gene regulation. Aberrant APA patterns have been associated with a wide range of diseases, from cancer to neuromuscular disorders. As such, a central question in the mRNA 3' processing field has been how polyadenylation sites (PAS) are recognized and how PAS selection can be regulated. The majority of mammalian PAS contain an AAUAAA hexamer and a U/UG-rich downstream element (DSE). According to the current model in the field, these key cis-elements are specifically recognized by CPSF160 of the CPSF complex and CstF64 of the CstF complex respectively. However, our published and preliminary data have challenged this model in several key aspects: 1) we have recently shown that the CPSF subunits CPSF30 and Wdr33, but not CPSF160 as was generally believed, directly bind to AAUAAA; 2) we have provided evidence that maintenance of the CPSF-RNA interaction specificity requires a novel proofreading factor(s) (see preliminary data); 3) we have demonstrated that the general 3' processing factor CstF64 in fact only binds to a subset of mammalian PAS, and we provided evidence that recognition of PAS with different sequence features requires distinct RNA-binding protein(s) (see preliminary data). These observations have not only significantly changed the current model for PAS recognition, but also revealed much greater complexity in mRNA 3' processing than previously appreciated. Here we propose to better define the molecular mechanisms of mammalian PAS recognition through an in-depth characterization of the key protein-RNA interactions involved. Accomplishing the proposed research will provide seminal insights into the fundamental mechanisms of mammalian mRNA 3' processing and its regulation. As aberrant PAS selection has been associated with a broad spectrum of human diseases, a better understanding of the mechanisms for PAS recognition may provide the foundation for the development of new therapeutics for these diseases.